1. Technical Field
The Wnt genes encode a large family of cysteine-rich secreted polypeptides that mediate diverse signalling processes. Aberrant activation of Wnt signaling plays important roles as a major driving force linked to developmental defects and tumorigenesis (Klaus and Birchmeier, 2008; Korinek et al., 1997; Morin et al., 1997; Willert et al., 2003). Wnt signaling pathways have been divided into two categories; one is the canonical Wnt/β-catenin signaling pathway and the other is the noncanonical Wnt/Ca2+ signaling pathway (Kühl et al., 2000; Liang et al., 2007; Liu et al., 2005). In the absence of Wnt activation, the level of β-catenin in the cytoplasm remains low due to the degradation of β-catenin by 26S proteasome after paired phosphorylation through casein kinase I (CKI) and glycogen synthase kinase-3β (GSK-3β) (Orford et al., 1997; Salic et al., 2000). The canonical Wnts bind to the Frizzled (Frz) family proteins and low-density lipoprotein receptor-related (LRP) 5 or 6, and this binding activates disheveled (Dvl) and inhibits the activity of GSK-3β; this inhibition results in the stabilization and subsequent translocation of β-catenin to the nucleus for the regulation of target gene expression with T-cell factor (TCF)/lymphoid enhancer factor (LEF) (Behrens et al., 1996; Giles et al., 2003; Molenaar et al., 1996; Moon et al., 2002).
Noncanonical Wnt signaling pathways affected by Wnt ligands such as Wnt5a have diverse and occasionally opposing roles (Slusarski et al., 1997; Tones et al., 1996). Noncanonical Wnts are both antagonistic and synergistic to canonical Wnt signalling pathway depending on their receptor context. Wnt5a-deficient mice show increased β-catenin signaling in the distal limb, indicating that Wnt5a is involved in the negative regulation of the Wnt/β-catenin signaling pathway (Nemeth et al., 2007). In contrast, Wnt5a has been shown to activate Wnt/β-catenin signaling in the presence of Frz4 and LRP5 (Mikels and Nusse, 2006). Given that the activation of the noncanonical Wnt signaling pathway results in intracellular Ca2+ release and activation of Ca2+ sensitive enzymes such as Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase C (PKC), the noncanonical Wnt pathways are apparently different from the canonical Wnt pathway.
2. Background Art
A mouse model of spontaneous intestinal tumorigenesis, designated APCmin/+, is widely used to explore Wnt/β-catenin signalling (Fodde et al., 1994; Shibata et al., 1997; Su et al., 1992). The genetic basis of familial associated polyposis (FAP) was mapped to the adenomatous polyposis coli (APC) gene, and germline and sporadic mutations in APC occur in most of FAP (Groden et al., 1991; Kinzler et al., 1991). APCmin/+ mice have a mutation in the APC gene causing hyperactivation of Wnt/β-catenin signaling and die within 6 months from severe intestinal tumor development. As Wnt/β-catenin signaling is crucial for the maintenance of cellular homeostasis, a variety of positive and negative cellular regulators have been identified using genetic, proteomic, and RNA interference-based screening approaches. Runx3 forms a ternary complex with TCF4/β-catenin and suppresses the DNA binding activity of TCF4/β-catenin (Ito et al., 2008). Wilms tumor suppressor WTX antagonizes Wnt/β-catenin signalling by promoting ubiquitination and degradation of β-catenin (Major et al., 2007). Recently, CDK8, a cyclin-dependent kinase member of the mediator complex, has been shown to be necessary for β-catenin-driven transcriptional activation (Firestein et al., 2008). Given that dysregulated transcriptional activity of β-catenin is crucial for colorectal tumorigenesis and progression, identification of genes that are responsible for genetic perturbations is important to explore complex malignant processes.
Members of the orphan nuclear receptor family play various roles in signal integration, including modulation of neurogenesis, homeostasis, and disease by regulating subsets of gene expression both positively and negatively (Blumberg and Evans, 1998; Giguère, 1999; Mangelsdorf et al., 1995). The retinoic acid-related orphan nuclear receptor (ROR)α is a member of the orphan nuclear receptor family for which no cognate ligands have been identified thus far (Giguère et al., 1994; Gold et al., 2003; Lau et al., 1994). Staggerer (sg) is a classical mutation of the RORα gene that blocks Purkinje cell differentiation, resulting in cerebellar hypoplasia and congenital ataxia (Hamilton et al., 1996). Sg mice exhibits phenotypes regarding lipid metabolism, bone metabolism, hyperinflammatory responses, and mainly cerebellar development and approximately 50% of the mice die shortly after weaning, which makes studying RORα function with Sg mice very difficult (Doulazmi et al., 2006).
Given that nuclear receptors function as potent regulators of normal physiology as well as pathologies such as cancer, the orphan nuclear receptors can functionally interact with potent oncogenic systems, for example, the Wnt and PKC signaling pathways (Peifer and Polakis, 2000). This interaction might elicit changes in oncogenesis and cellular adhesion (Polakis, 2000; van de Wetering et al., 2002). Compared to other classes of nuclear receptors, the function and related signaling pathways for the orphan nuclear receptor RORα have not yet been studied extensively.
Thus, the present inventors identified a critical role of RORα at the crossroads between the canonical and the noncanonical Wnt signalling pathways in attenuating β-catenin transcriptional activity in a phosphorylation-dependent manner in colon cancer, based on cell culture, colorectal carcinoma tissues, and mouse cancel model studies. Biochemical purification of RORα containing complex identifies β-catenin as a component, providing a novel link between RORα and Wnt signaling pathway. Analysis of RORα interactions with β-catenin reveals that the RORα-mediated inhibition of Wnt/β-catenin signalling requires Wnt5a/PKCα induced phosphorylation on serine residue 35 of RORα, and the binding of RORα to β-catenin is triggered and enhanced by phosphorylation of RORα. Intriguingly, reduction of phosphorylation of RORα concomitant with downregulation of PKC is correlated with activation of Wnt target genes and tumor progression in colorectal carcinoma tissues. The present inventors invented the present invention by revealing of role of RORα in transrepression of the Wnt/β-catenin signalling pathway, thereby regulating cell proliferation and tumor progression in a pathophysiological model.